We have already fine-mapped microbial QTL down to an average of 4 Mb (e.g. Neisseria). To further reduce these fragments we will sequence the microbiota from additional AIL mice. Using this approach we are likely to reduce the QTLs to 1-2 Mb. We will then proceed to identify the putative candidate genes by targeted sequencing of the entire fragments in the parental strains. The expression of candidate genes will be examined in relevant tissues (immune cells, skin and gut epithelium).  Depending on the gene in question, strategies to inactivate or overexpress it will be implemented.

• We will culture at least two OTUs with confirmed association to host genome variants in G4/G16. To further characterize the strains we will fully sequence their genomes using the Illumina MiSeq platform (see our published report Wang J et al. Genome Announc. 2014;2(1). pii: e01148-13).
• To confirm their role in disease, we will expose germ-free mice to bacterial communities differing in the presence/absence abundance of candidate bacteria, followed by disease induction.  The same will be applied to knockout identified relevant genes. To confirm interaction, we will perform cross-fostering experiments under germ free conditions.
• To identify the potential pathways involved, we will perform a series of co-culture experiments involving immune cells isolated from control or mutant mice with and without candidate bacteria. The effect on cytokine production, cell death, activation, proliferation and gene expression will be evaluated.